The objective of this project is to elucidate the molecular mechanisms by which interferons (IFN) regulate the expression of a number of specific genes in human fibroblasts. It has been demonstrated that IFN treatment induces the synthesis of several new proteins in these cells and induction of these proteins parallels induction of the antiviral state. Our preliminary results indicate that synthesis of these proteins in IFN-treated cells is regulated both at transcriptional level and at translational level. Moreover, the nature of this regulation is different in IFNalpha-\and IFN-gamma-treated cells. In this project we will isolate and characterize cDNA clones corresponding to IFN-induced mRNAs. We will use these cloned cDNAs as hybridization probes for identifying different IFN-inducible mRNAs and for studying the regulation of synthesis and turnover of these mRNAs. We also will use the cDNA clones to screen a human genomic library for isolation of the corresponding genes. Once the genomic clones are isolated they will be characterized by restriction enzyme-mapping. Finally, we will introduce the isolated IFN-inducible genes into heterologous cells and examine their expression. This project will provide us with reagents and assay systems necessary for achieving our long-term objectives of understanding the nature of the molecular signals that regulate the expression of these IFN-inducible genes and of correlating the induction of a specific gene with a specific IFN-inducible biological phenotype. Information obtained from this system will also be valuable for understanding the modes of regulation of eukaryotic gene expression in general. Efforts to isolate IFN-inducible cDNA clones will be continued. A cDNA library has been prepared using mRNA from IFN-treated cells. The clones in this library will be differentially screened using cDNAs made against mRNAs from IFN-treated and untreated cells. Potentially interesting clones will be picked and rescreened. Plasmid DNA will be isolated from candidate clones, nick translated, and hybridized against mRNAs from IFN-treated and untreated cells. Positive clones will be classified into independent families by virtue of cross-hybridization data. These clones will be used as hybridization probes for studying the synthesis and turnover of the corresponding mRNAs. One IFN-inducible mRNA whose level we will be able to measure in the immediate future is the 2-5 (A) synthetase mRNA. We are currently standardizing the conditions for using a synthetic oligonucleotide probe for this measurement. Once these methods are optimized, we will study the regulation of synthesis of this mRNA in response to IFN-treatment of a variety of cells. We have isolated three classes of cDNA clones related to 2-5 (A) synthetase mRNAs. They hybridize with mRNAs of about 2kb and 5kb lengths which are induced by IFN. We have also isolated a cDNA clone corresponding to another IFN-inducible mRNA encoding a 56kd protein. We have initiated studies on regulation of synthesis and turnover of p56 mRNA by IFN. Cytoplasmic level of this mRNA increases to the maximum after 6 hr of IFN treatment. The level then declines rapidly even if IFN is present continuously in the culture medium. This rapid turnover could be prevented by treating the cells with actinomycin D or cycloheximide after p56 mRMA had been induced. It appears, therefore, that IFN not only induces the synthesis of p56 mRNA but also induces the synthesis of a regulatory protein which causes rapid tumors of p56 mRNA. (N)